Promoting axon regeneration in the adult CNS through control of protein translation

ABSTRACT

Survival of, or axon regeneration in a lesioned mature central nervous system (CNS) neuron is promoted by (a) contacting the neuron with a therapeutically effective amount of an exogenous activator of protein translation; and (b) detecting the resultant promotion of the survival of, or axon regeneration in the neuron.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/729,091, filed on Dec. 28, 2012, issued as U.S.Pat. No. 8,728,756, which is a continuation application of U.S. patentapplication Ser. No. 12/479,805, filed on Jun. 6, 2009, issued as U.S.Pat. No. 8,367,352 B2 on Feb. 5, 2013, which claims benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 61/059,568, filedJun. 6, 2008, the contents of which are incorporated herein by referencein their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant PO30-HD18655awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The field of the invention is activating protein synthesis to promoteregeneration of a lesioned CNS axon and compensatory regrowth of aspared axon in the mature CNS.

Axon regeneration failure following injury in the adult mammalian CNShas been attributed mainly to two properties of the adult CNS, namelythe inhibitory extrinsic environment and diminished intrinsicregenerative capacity of mature CNS neurons (1-5). Numerous studies onthe non-permissive environment have led to the identification of anumber of molecular players and signaling pathways involved in limitingaxon regrowth. While these mechanisms clearly represent importantextrinsic barriers for axon regeneration, the strategies to neutralizethese inhibitory activities only allowed a limited degree of axonregeneration in vivo (6, 7). Indeed, a permissive environment, such as asciatic nerve graft transplanted to the lesion site, allows a smallpercentage of injured adult axons to regenerate (5, 8). These resultsindicate that neutralizing inhibitory activity is not sufficient andtherefore other mechanisms, such as those controlling the intrinsicaxonal regenerative potential of neurons, may play an important role inaxon regeneration.

In contrast to the axon growth during embryonic development, little isknown about the molecular mechanisms that control the intrinsicregenerative ability of adult CNS neurons (1, 3-5, 9). It is alsounknown whether similar or different mechanisms operate axon growthduring development and axon regeneration following injury and whataccounts for the decreased ability of axon growth over the course ofdevelopment. Both transient and sustained axon sprouting has beendocumented in the adult CNS as the anatomical basis of structuralplasticity in response to activity deprivation (10). The reason for thisreorganization of axons as a compensatory mechanism, in the face offailure to regenerate injured axons, is also unclear. A potential hintto these questions comes from the evolutionarily conserved molecularpathways that control cellular growth and size. It is believed that formost of cell types, specific mechanisms are necessary in preventingcellular overgrowth upon the completion of development (11). Since manyof these molecules are often expressed in post-mitotic mature neurons,we hypothesized that these pathways may contribute to the diminishedregenerative ability in adult CNS neurons.

By testing different pathways involved in cell growth control in anoptic nerve injury model, we show that inhibiting PTEN (phosphatase andtensin homolog), a negative regulator of the mammalian target ofrapamycin (mTOR) pathway, in the adult retinal ganglion cells (RGCs)promotes striking axon regeneration. Further studies revealed a two-stepsuppression of mTOR signaling in adult CNS neurons: first bydevelopmental maturation and second by axotomy-triggered stressresponse. Such inhibition of mTOR activity and subsequent impairment innew protein synthesis ability contributes to their inability toregenerate injured axons. Reactivating this pathway by inhibitingtuberous sclerosis complex 1 (TSC1), another negative regulator of themTOR pathway, also leads to extensive long-distance axon regeneration.Our work shows that general growth control pathways regulate axonregenerative abilities in neurons, thereby providing new strategies topromote axon regeneration after CNS injury such as spinal cord injury,stroke, traumatic brain injury and glaucoma.

Subsequent to our priority filing date, Nakashima et al. (J Neurosci(2008 Jul. 16) 28(29):7293-303) reported small-molecule protein tyrosinephosphatase inhibition using potassiumbisperoxo(1,10-phenanthroline)oxovanadate (V) as a neuroprotectivetreatment after spinal cord injury in adult rats.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for promoting survivalof, or axon regrowth (regeneration and sprouting) in a lesioned maturecentral nervous system (CNS) neuron in situ. The general methodscomprise: (a) contacting the neuron with a therapeutically effectiveamount of an exogenous activator of protein translation in the neuron,and thereby promote survival of, or axon regeneration in the neuron; and(b) detecting the resultant promotion of the survival of, or axonregeneration in the neuron.

In particular embodiments, the activator of protein translation is: (a)a mTOR pathway activator; (b) a PTEN inhibitor; (c) a TSC1/2 inhibitor;(d) an Akt activator; (e) a Ras/MEK pathway activator; or (f) a PRAS40inhibitor.

In particular embodiments, the activator of is a PTEN inhibitor such as(a) potassium bisperoxo(bipyridine)oxovanadate (V) (bpV(bipy)); (b)dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (V)(bpV(HOpic)); (c) potassium bisperoxo(1,10-phenanthroline)oxovanadate(V), (bpV(phen)); or (d) dipotassium bisperoxo(picolinato)oxovanadate(V), (bpV(pic)).

In various embodiments, the lesion results from a traumatic injury,traumatic brain injury, a stroke, an acute spinal cord injury, or CNSdegeneration.

In specific embodiments, the lesioned axon is in the optic nerve, or isa CNS axon of a sensory neuron, or is in the spinal cord.

In a specific embodiment, the lesioned axon is in the spinal cord of apatient, and the inhibitor is intrathecally administered to the patient.

In various embodiments, the axon is a CNS axon of a sensory neuron, or aCNS axon of a cerebellar granule neuron.

The detecting step may be effected by an indirect or direct assay ofaxon regeneration.

In various embodiments, the inhibitor is administered intravenously,intrathecally, ocularly, or locally at the neuron.

In a particular embodiment, the general method further comprise anantecedent step of determining that the neuron is lesioned, and hasaxotomy-induced stress and/or pathology-induced down-regulation ofprotein translation.

In a particular embodiment, the activator of is a PTEN inhibitor, thelesioned axon is in the optic nerve, and the inhibitor is administeredocularly.

In another aspect, the invention provides compositions specificallyadapted to the subject methods, such as a device for promoting survivalof, or axon regeneration in a lesioned mature central nervous system(CNS) neuron in situ determined to have axotomy-induced stress and/orpathology-induced down-regulation of protein translation, comprising areservoir loaded with a premeasured and contained amount of atherapeutically effective amount of an activator of protein translationin the neuron, and specifically adapted for implementing the subjectmethods.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The target neuron is lesioned and has axotomy-induced stress and/orpathology-induced down-regulation of protein translation, which may bedetected directly, indirectly, or inferred. In particular examples thelesioned axon is a CNS axon of a dorsal root ganglion (DRG) sensoryneuron, a cerebellar granule neuron, or an ocular neuron. The mature(i.e. terminally-differentiated, non-embryonic) neuron may be in vitroor in situ in a subject or patient. In specific embodiments, the subjectis a mammal (e.g. human, companion animal, livestock animal, rodent orprimate animal model for neurodegeneration or CNS injury, etc.).

The lesion can result from traumatic injury, optic nerve injury ordisorder, brain injury, stroke, chronic neurodegeneration such as causedby neurotoxicity or a neurological disease or disorder (e.g.Huntington's disease, Parkinson's disease, Alzheimer's disease, multiplesystem atrophy (MSA), etc.).

In one embodiment, the activator is used to treat an ocular injury ordisorder (e.g. toxic amblyopia, optic atrophy, higher visual pathwaylesions, disorders of ocular motility, third cranial nerve palsies,fourth cranial nerve palsies, sixth cranial nerve palsies, internuclearophthalmoplegia, gaze palsies, eye damage from free radicals, etc.), oran optic neuropathy (e.g. ischemic optic neuropathies, toxic opticneuropathies, ocular ischemic syndrome, optic nerve inflammation,infection of the optic nerve, optic neuritis, optic neuropathy,papilledema, papillitis, retrobulbar neuritis, commotio retinae,glaucoma, macular degeneration, retinitis pigmentosa, retinaldetachment, retinal tears or holes, diabetic retinopathy, iatrogenicretinopathy, optic nerve drusen, etc.).

In a particular embodiment, the lesion results from acute or traumaticinjury such as caused by contusion, laceration, acute spinal cordinjury, etc. In specific embodiments, the lesioned CNS axon is in CNSwhite matter, particularly white matter that has been subjected totraumatic injury. In certain embodiments, the contacting step isinitiated within 96, 72, 48, 24, or 12 hours of formation of the lesion.In various embodiments, the contacting step is initiated, and/ortreatment is continued, more than 5, 7, 14, 30, or 60 days afterformation of the lesion.

The activator can be administered to the injured neuron in combinationwith, or prior or subsequent to, other treatments such as the use ofanti-inflammatory or anti-scarring agents, growth or trophic factors,etc. In a specific embodiment, the lesion results from acute spinal cordinjury and the method additionally comprises contacting the neuron withmethylprednisolone sufficient to reduce inflammation of the spinal cord.In various other embodiments, the activator is administered incombination with trophic and/or growth factors such as NT-3 (Piantino etal, Exp Neurol. 2006 October; 201(2):359-67), inosine (Chen et al, ProcNatl Acad Sci USA. (2002) 99:9031-6; U.S. Pat. No. 6,551,612 toBenowitz; U.S. Pat. No. 6,440,455 to Benowitz; and US Pat Publ20050277614 to Benowitz), oncomodulin (Yin et al, Nat. Neurosci. (2006)9:843-52.; US Pat Publ 20050054558 to Benowitz; US Pat Publ 20050059594to Benowitz; and U.S. Pat. No. 6,855,690 to Benowitz), etc.

We have documented the suitability in the subject methods andcompositions of a wide variety of activators of protein synthesis, andalternative suitable activators are readily identified using the assaysdescribed and exemplified herein. In general, the activator increasesthe effective amount of one or more positive regulators of proteinsynthesis (such as RheB1, Akt, Ras, etc.), or inhibits the effectiveamount of one or more negative regulators of protein synthesis (such asPTEN, TSC1, TSC2, PRAS40, etc.). Increasing positive regulators can beeffected by simply employing the positive regulator or a more activevariant thereof as the activator. Inhibiting negative regulators can beeffected at the level of transcription (e.g. using specific siRNA), orby targeting the negative regulator with specific pharmacologicalinhibitors. Exemplary suitable activators include: mTOR pathwayactivators, such as active RheB1; PTEN inhibitors, such as establishedvanadium-based PTEN inhibitors or siRNA; TSC1/2 inhibitors, such assiRNA for TSC2 or TSC1; Akt activators, such as active Akt andmenadione; Ras/MEK pathway activators, such as active Ras; and PRAS40inhibitors, such as siRNA. Alternative, suitable activators of proteinsynthesis are readily identified, confirmed and characterized using theassays and protocols disclosed herein.

In particular embodiments, the activator is a PTEN inhibitor,particularly a vanadium-based PTEN inhibitor, such as sold commerciallyby Calbiochem, EMD/Merck, including (a) potassiumbisperoxo(bipyridine)oxovanadate (V) (bpV(bipy)); (b) dipotassiumbisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (V) (bpV(HOpic)); (c)potassium bisperoxo(1,10-phenanthroline)oxovanadate (V), (bpV(phen));and (d) dipotassium bisperoxo(picolinato)oxovanadate (V), (bpV(pic)).Alternative, suitable PTEN inhibitors include PTEN inhibitor compoundsof formulas I-XIV as described in WO2005/097119; vanadium-based PTENinhibitors described in US20070292532 and by Rosivatz et al. 2006 (ACSChem. Bio1.1 (12) 780-790); the 1,4-naphthoquinone derivative, shikonin,described by Nigorikawa et al. (Mol Pharmacol 70:1143-1149, 2006); andmenadione (vitamin K3) as described by Yoshikawa et al., Biochim BiophysActa. 2007 April; 1770(4):687-93. PTEN inhibition assays for generalscreening (to identify and confirm alternative, suitable inhibitors) andIC50 determinations are known in the art, e.g. WO 2005/097119; see alsoExample 2, below.

Suitable PTEN inhibitors are also described in WO 2007/0203098,including all recited genera, subgenera and species disclosed and asdescribed therein including:

I) Ascorbic Acid-Based PTEN Inhibitors

wherein,R1 represents H, C1-C3 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR3,(CH₂)_(n)XCOR3, (CH₂)_(n)COR3, (CH₂)_(n)SO₂R3, (CH₂)nXR3, (CH₂)nSO₂X—R3,(CH₂)_(n)XSO₂R3, (CH₂)_(n)NR3R4, or (CH₂)_(n)CO(CH₂)_(m)XR3;R2 represents H, C1-C3 alkyl, aryl, alkylaryl, (CH₂)_(n)COX—R3,(CH₂)_(n)XCOR3, (CH₂)_(n)COR3, (CH₂)_(n)SO₂R3, (CH₂)_(n)XR3,(CH₂)nSO₂X—R3, (CH₂)_(n)XSO₂R3, (CH₂)_(n)NR3R4, or(CH₂)_(n)CO(CH₂)_(m)XR3;R3, R5 and R6 independently are H, C1-C4 alkyl, aryl or alkylaryl;R4 represents H, C1-C4 alkyl, aryl, alkylaryl, NHSO₂R5, NHCO₂R5, orNR5R6;m=O to 3;n=O to 3; andX represents O or NR4.Compounds of Formula I and Ia may have ester linkages at either R1 orR2.

II) 1,2,3-triazole PTEN Inhibitors (Such as Described in WO02/32896)

wherein,R1 represents H, C1-C4 alkyl, aryl, alkylaryl, COXR2, COR2, SO₂XR2,SO₂R2;R2 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR4,(CH₂)_(n)XCOR4, (CH₂)_(n)X R4,(CH₂)_(n)SO₂XR4, (CH₂)_(n)XSO₂R4, NHSO₂R4, NHCOR4, NHCO₂R4, NHCOCO₂R4,or NR4R5;R3 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR4,(CH₂)_(n)XCOR4, (CH₂)_(n)X R4, (CH₂)_(n)SO₂XR4, (CH₂)_(n)XSO₂R4,NHSO₂R4, NHCOR4, NHCO₂R4, NHCOCO₂R4, or NR4R5;R4 represents H, C1-C4 alkyl, aryl, or alkylaryl;R5 represents H, C1-C4 alkyl, aryl, alkylaryl,NHSO₂R6, NHCOR6, NHCO₂R6, NR6R7, or N═C(R6R7);R6 represents H, C1-C4 alkyl, aryl, or alkylaryl;R7 represents H, C1-C4 alkyl, aryl, or alkylaryl;n=0-3; andX represents O or NR5.

The inhibitors of Formula II include:

wherein,R8 represents (CH₂)_(n)XR4, or (CH₂)_(n)SR4;R9 represents NHNHSO₂ aryl, NHNHCO-aryl, or NHN═C(R6R7); andR10 represents H, C1-C4 alkyl, aryl, alkylaryl, SO₂R6, COR6, or CO₂R6.

III) Diamide PTEN Inhibitors

wherein,A is a five or six member ring;R1 represents H, C1-C3 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR3,(CH₂)_(n)XCOR3, (CH₂)_(n)COR3, (CH₂)SO₂R3, (CH₂)_(n)XR3, (CH₂)SO₂XR3,(CH₂)_(n)XSO₂R3, NHSO₂R3, NHCO₂R3,NHCOR3, NHCO₂R3, NHCOCO₂R3, NR3R4, or (CH₂)_(n)CO(CH₂)_(m)XR3;R2 represents H, C1-C3 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR3,(CH₂)_(n)XCOR3, (CH₂)_(n)COR3, (CH₂)_(n)SO₂R3, (CH₂)_(n)XR3,(CH₂)_(n)SO₂XR3, (CH₂)_(n)XSO₂R3; NHSO₂R3, NHCO₂R3,NHCOR3, NHCO₂R3, NHCOCO₂R3, NR3R4, or (CH₂)_(n)CO(CH₂)_(m)XR3;R3 represents H, C1-C4 alkyl, aryl, or alkylaryl;R4 represents H, C1-C4 alkyl, aryl, alkylaryl, NHSO₂R5, NHCO₂R5, orNR5R6;R5 represents H, C1-C4 alkyl, aryl, or alkylaryl;R6 represents H, C1-C4 alkyl, aryl, or alkylaryl;n=0-3;m=0-3; andX represents O, or NR4.

Ring A may be saturated, unsaturated, or aromatic, and may optionallycomprise N and O. Preferred compounds of formula III are those whereinring A is selected from heterocyclic ring systems, especially vicinallysubstituted pyridines, pyrimidines, furazans, imidazoles, pyrrazoles,furaus, thiazoles, and oxazoles, as well as their saturated analogs;other preferred inhibitors of formula III are those wherein ring Acomprises an all carbon aromatic rings, such as substituted andunsubstituted phenyl, and their saturated analogs.

The inhibitors of Formula III may comprise a ring A selected from thefollowing:

The inhibitors of Formula III comprising a ring A selected from IIIA,IIIB, IIIC, IIID, IIIE may further comprise:

R1 represents H, C1-C3 alkyl, aryl, alkylaryl, (CH₂)_(n)COR3, or(CH₂)_(n)SO₂R3;

R3 represents H, C1-C4 alkyl, aryl, or alkylaryl;

R7 represents H, C1-C4 alkyl, halogens, NO₂, CF₃, aryl, carboxylate,aryloxy, amino, alkylamino, cyano, isocyanate, alkoxycarbonyl, orhaloalkyl;

R8 represents H, C1-C4 alkyl, halogens, NO₂, CF₃, aryl, carboxylate,aryloxy, amino, alkylamino, cyano, isocyanate, alkoxycarbonyl, orhaloalkyl; and

m=1,2,3.

In particular embodiments, alkylaryl is selected from Formula IIIF orIIIG:

The inhibitors of Formula III may also be of the formula:

wherein,A is a five or six member ring;R9 represents H, C1-C3 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR3,(CH₂)_(n)XCOR3, (CH₂)_(n)COR3, CH₂(CH₂)_(n)SO₂R3, CH₂(CH₂)_(n)XR3,CH₂(CH₂)_(n)SO₂XR3, or CH₂(CH₂)_(n)XSO₂R3;R10 represents H, C1-C3 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR3,(CH₂)_(n)XCOR3, (CH₂)_(n)COR3, CH₂(CH₂)_(n)SO₂R3, CH₂(CH₂)_(n)XR3,CH₂(CH₂)_(n)SO₂XR3, or CH₂(CH₂)_(n)XSO₂R3; andR3, X, and n are as described for Formula III.

Ring A of inhibitors IIIH and IIIJ may be saturated, unsaturated oraromatic, and may optional be substituted with C and N.

IV) Aryl Imidazole Carbonyl PTEN Inhibitors

wherein,R1 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR3,(CH₂)_(m)XCOR3, (CH₂)_(m)XR3, (CH₂)_(n)COR3, (CH₂)_(n)SO₂XR3, or(CH₂)_(m)XSO₂R3;R2 represents H, C1-C4 alkyl, aryl, or alkylaryl;R3 represents H, C1-C3 alkyl, aryl, or alkylaryl;R4 represents H, C1-C4 alkyl, aryl, alkylaryl, NHSO₂R5, NHCO₂R5,N═C(R5R6), or NR5R6;R5 represents H, C1-C4 alkyl, aryl, or alkylaryl;R6 represents H, C1-C4 alkyl, aryl, or alkylaryl;m=1-3;n=0-3; andX represents 0, NR4.

Compounds of formula IV may be of the formula:

wherein, R7 represents XR4.

Compounds of formula IV may also be of the formula:

whereinR8 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COX—R3,(CH₂)_(n)XCOR3, (CH₂)_(n)X—R3, (CH₂)_(n)COR3, (CH₂)_(n)SO₂XR3, or(CH₂)_(n)XSO₂R3; andR9 represents H, C1-C4 alkyl, aryl, alkylaryl.

Compounds of Formula IVB may also be selected:

V) Polyamide PTEN Inhibitors

VI) Commercial PTEN Inhibitors

-   1. Deltamethrin; (S)-a-Cyano-3-phenoxybenzyl(1R)-cis-3-(2,2    dibromovinyl)-2,2-dimethylcyclopropanecarboxylate-   2. Alendronate, Sodium, Trihydrate-   3.    N-(9,10-Dioxo-9,10-dihydrophenanthren-2-yl)-2,2-dimethylpropionamide-   4. 5-Benzyl-3furylmethyl (1R,S)-cis,trans-chrysanthemate-   5. Suramin, Sodium Salt;    8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-methyl-3,1-phenylene)carbonylimino]]bis-,    hexasodium salt-   6. 4-Methoxyphenacyl Bromide-   7. 1,4-Dimethylendothall;    1,4-Dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic Acid-   8. Cantharidic Acid;    2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3dicarboxylic acid-   9. Sodium Stibogluconate; Antimony Sodium Gluconate-   10. 3,4-Dephostatin, Ethyl--   11. Fenvalerate;    a-Cyano-3-phenoxybenzyl-a(4-chlorophenyl)isovalerate-   12. α-Naphthyl Acid Phosphate, Monosodium Salt-   13. β-Glycerophosphate, Disodium Salt, Pentahydrate-   14. Endothall; 7-Oxabicyclo[2.2.1]heptane-2,3dicarboxylic Acid-   15. Cypermethrin;    (R,S)-α-Cyano-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate;    (1R)—(R)cyano(3-phenoxyphenyl)methyl    3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate.

VII) 1,10-phenanthroline-5,6-dione PTEN Inhibitors

wherein,R1 represents 0, C1-C4 alkyl, (CH₂)_(n)COXR2, (CH₂)_(n)XCOR2,(CH₂)_(n)XR2, (CH₂)_(n)COR2, (CH₂)SO₂XR2, (CH₂)_(n)XSO₂R2, or(CH₂)_(n)SO₂R2;R2 represents H, C1-C4 alkyl, aryl, alkylaryl, NHSO₂R4, NHCOR4, NHCO₂R4,NHCOCO₂R4, or NR4R5;R3 represents H, C1-C4 alkyl, aryl, alkylaryl, NHSO₂R4, NHCOR4, NHCO₂R4,NHCOCO₂R4, or NR4R5;R4 represents H, C1-C4 alkyl, aryl, or alkylaryl;R5 represents H, C1-C4 alkyl, aryl, or alkylaryl;R6 at each occurrence is independently selected from hydrogen, halogen,NO₂, NR4R10, C1-C4 alkyl, NH(CH₂)_(p)CO(CH₂)_(q)XR2, (CH₂)_(p)COXR2,(CH₂)_(p)XCOR2, (CH₂)_(p)XR2, (CH₂)pCOR2, (CH₂)_(p)SO₂XR2, or(CH₂)_(p)XSO₂R2;R7 represents H, C1-C4 alkyl, aryl, alkylaryl, SO₂R4, NHSO₂R4, NHCO₂R4,or NR8R9;R8 represents independently H, C1-C4 alkyl, aryl, alkylaryl,(CH₂)_(n)COXR2, or (CH₂)_(n)XR2;R9 represents independently H, C1-C4 alkyl, aryl, alkylaryl,(CH₂)_(n)COXR2, (CH₂)_(n)XR2, (CH₂)_(p)COXR2, (CH₂)_(p)XCOR2, (CH₂)—XR2,(CH₂)_(p)COR2, (CH₂)_(p)SO₂XR2, (CH₂)_(p)XSO₂R, or (CH₂)_(p)SO₂R2;R10 represents H, C1-C4 alkyl R7=H, C1-C4 alkyl, aryl, alkylaryl, SO₂R4,NHSO₂R4, NHCO₂R4, or NR8R9;m represents independently 0 or 1;n=1-5;p=0-5;q=0-5;X represents O or NR3; andZ═O or NR7.The nitrogen in the ring of compound of Formula VII may be neutral. Thenitrogen may also be charged when bound to an R1 group (quaternary salt)in the case where at least one m=1.

The inhibitor may also be selected from:

VIII) Substituted phenathrene-9-10-dione PTEN Inhibitors

wherein,R1 represents H, NO₂, NR5R6, halogen, cyano, alkyl, alkylaryl, carbonyl,carboxy, COR2, or CONR5R6;R2 and R3 represent independently H, C1-C4 alkyl, aryl, or alkylaryl;R4 represents H, C1-C4 alkyl, aryl, alkylaryl, SO₂—R2, NHSO₂R2, NHCOR2,NHCO₂R2, N═CR2R3, or NR5R6;R5 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR2,(CH₂)_(n)XR2, (CH₂)_(n)CO(CH₂)_(m)XR2, SO₂R2, (CH₂)_(n)CO(CH₂)_(n)COXR2,or (CH₂)_(n)COR2;R6 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COX—R2,(CH₂)_(n)XR2, (CH₂)_(n)CO(CH₂)_(m)XR2, SO₂R2, (CH₂)_(n)CO(CH₂)_(n)COXR2,or (CH₂)_(n)COR2;m=0-3;n=0-3; andX represents CR2R3, O, NR4.

The inhibitors of Formula VIII may be of the formula:

IX) Isatin PTEN Inhibitors

wherein,R1 represents H, NO₂, NR5R6, halogen, cyano, alkyl, alkylaryl, carbonyl,carboxy, COR₂, CONR5R6, SO₃R2, or SO₂NR2R3;R2 and R3 represent independently H, C1-C4 alkyl, aryl, or alkylaryl;R4 represents H, C1-C4 alkyl, aryl, alkylaryl, SO₂—R2, NHSO₂R2, NHCOR2,NHCO₂R2,N═CR2R3, or NR5R6;R5 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COX—R₂,(CH₂)_(n)X—R₂, (CH₂)_(n)CO(CH₂)_(m)XR2, SO₂R2,(CH₂)_(n)CO(CH₂)_(n)COXR2, or (CH₂)_(n)COR2;R6 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COX—R2,(CH₂)_(n)X—R2 (CH₂)_(n)CO(CH₂)═XR2, SO₂R2, (CH₂)_(n)CO(CH₂)_(n)COXR2, or(CH₂)_(n)COR2;m=0-3;n=0-3; andX represents CR2R3, O, NR4.

The inhibitors of Formula IX may be selected from:

X) Substituted phenanthren-9-ol PTEN Inhibitors

R1 represents H, N02, NR5R6, halogen, cyano, alkyl, alkylaryl, carbonyl,carboxy, COR2, or CONR5R6;R2 and R3 represent independently H, C1-C4 alkyl, aryl, or alkylaryl;R4 represents H, C1-C4 alkyl, aryl, alkylaryl, SO₂—R2, NHSO₂R2, NHCOR2,NHCO₂R2, N═CR2R3, or NR5R6;R5 represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COXR2,(CH₂)_(n)X—R2, (CH₂)_(n)CO(CH₂)_(m)XR2, SO₂R2,(CH₂)_(n)CO(CH₂)_(n)COXR2, or (CH₂)_(n)COR2;RO represents H, C1-C4 alkyl, aryl, alkylaryl, (CH₂)_(n)COX—R2,CCH₂)_(n)X—R2, (CH₂)_(n)CO(CH₂)_(m)XR2, SO₂R2,(CH₂)_(n)CO(CH₂)_(n)COXR2, (CH₂)_(n)COR2;m=0-3;n=0-3; andX represents CR2R3, O, NR4.

XI) Substituted naphthalene-1,2-dione PTEN Inhibitors

wherein,R1 represents independently chosen from H, NO₂, NR3R4, halogen, cyano,alkyl, alkylaryl, carbonyl, carboxy, (CH₂)_(n)COXR3, COR2, SO₃—R2,SO₂N—R3R4, NHSO₂—R3, NHCO₂R3, NHCOR3, NHCOCO₂R2, NR3R4, or CONR3R4;R2 represents H, C1-C4 alkyl, aryl, or alkylaryl;R3 and R4 represent independently H, C1-C4 alkyl, aryl, alkylaryl,(CH₂)_(n)COXR2, (CH₂)_(n)CO(CH₂)_(m)XR2, or (CH₂)_(n)OR2;m=0-3;n=0-3; andX represents O, NR2.

The inhibitors of Formula XI may be selected from:

XII) Substituted naphthalene-1,4-dione PTEN Inhibitors

whereinR1 represents H, NO₂, NR3R4, halogen, cyano, alkyl, alkylaryl, carbonyl,carboxy, (CH₂)_(n)COXR3, COR2, SO₃R2, SO₂N—R3R4, NHSO₂—R3, NHCO₂R3,NHCOR3, NHCOCO₂R2, NR3R4, or CON—R3R4;R2 represents H, C1-C4 alkyl, aryl, or alkylaryl;R3 and R4 represents independently H, C1-C4 alkyl, aryl, alkylaryl,(CH₂)_(n)COXR2, (CH₂)_(n)CO(CH₂)_(m)XR2, or (CH₂)_(n)OR2;m=0-3;n=0-3; andX represents O, NR2.

XIII) Vanadate-Based PTEN Inhibitors

-   1. Potassium Bisperoxo(bipyridine)oxovanadate (V)-   2. Dipotassium Bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate    (V)-   3. Dipotassium Bisperoxo(picolinato)oxovanadate (V)-   4. Monoperoxo(picolinato)oxovanadate (V)-   5. Potassiun Bisperoxo(1,10-phenanthroline)oxovanadate (V)-   6. bis(N,N-Dimethylhydroxamido)hydroxooxovanadate

XIV) T1-Loop Binding Element Containing PTEN Inhibitors

The PTEN inhibitors may contain a group that exists at physiological pHin significantly anionic form, such as at least 5% of the molecularspecies at pH of 7.4 are anionic charged. Such anionic groups can bindto PTEN in the T1 loop of the peptide structure in solution.

Representative examples of such groups include:

wherein, R is independently chosen from H, OH, O-alkyl, alkyl, SH,S-alkyl, NH2, NH-alkyl, N-(alkyl)2 where alkyl is a small, C1-C4 alkylmoiety. The dashed lines represent the connection to the formulas of thecompounds described for Formulas I through XIII above. The groups may befurther evaluated in silico for their ability to fill the T1 loop spaceby standard molecular docking procedures. Such TI-Ioop binding groupsmay be incorporated into compounds of Formula I-XIII. Incorporation ofthe groups may impart selectivity of the molecules to inhibition ofPTEN. Preparation of groups XIVa-XIVd are well established in theliterature. Compounds of Formula XIVe may be prepared by methodsdisclosed in Wilson et al., Bioorganic & Medicinal Chemistry Letters,vol 6, No. 9, pp 1043-1046, 1996. Incorporation of these groups into theFormulas I-XIII is by standard synthetic methods easily attainable bythose skilled in the art. Examples of such incorporation by simplyutilizing appropriate starting materials is illustrated by theconversion of 7-9 to one incorporating the above groups, e.g.:

The activator is contacted with the neuron using a suitable drugdelivery method and treatment protocol sufficient to promoteregeneration of the axon. For in vitro methods, the activator is addedto the culture medium, usually at nanomolar or micromolarconcentrations. For in situ applications, the activator can beadministered orally, by intravenous (i.v.) bolus, by i.v. infusion,subcutaneously, intramuscularly, ocularly (intraocularly, periocularly,retrobulbarly, intravitreally, subconjunctivally, topically, by subtenonadministration, etc.), intracranially, intraperitoneally,intraventricularly, intrathecally, by epidural, etc.

Depending on the intended route of delivery, the compositions may beadministered in one or more dosage form(s) (e.g. liquid, ointment,solution, suspension, emulsion, tablet, capsule, caplet, lozenge,powder, granules, cachets, douche, suppository, cream, mist, eye drops,gel, inhalant, patch, implant, injectable, infusion, etc.). The dosageforms may include a variety of other ingredients, including binders,solvents, bulking agents, plasticizers etc.

In a specific embodiment, the activator is contacted with the neuronusing an implantable device that contains the activator and that isspecifically adapted for delivery to a CNS axon of neuron. Examples ofdevices include solid or semi-solid devices such as controlled releasebiodegradable matrices, fibers, pumps, stents, adsorbable gelatin (e.g.GELFOAM™), etc. The device may be loaded with premeasured, discrete andcontained amounts of the activator sufficient to promote regeneration ofthe axon. In a particular embodiment, the device provides continuouscontact of the neuron with the activator at nanomolar or micromolarconcentrations, preferably for at least 2, 5, or 10 days.

The subject methods typically comprise the further step of detecting aresultant regeneration of the axon. For in vitro applications, axonalregeneration may be detected by any routinely used method to assay axonregeneration such as a neurite outgrowth assay. For in situapplications, axonal regeneration can be detected directly using imagingmethodologies such as MRI, or indirectly or inferentially, such as byneurological examination showing improvement in the targeted neuralfunction. The detecting step may occur at any time point afterinitiation of the treatment, e.g. at least one day, one week, one month,three months, six months, etc. after initiation of treatment. In certainembodiments, the detecting step will comprise an initial neurologicalexamination and a subsequent neurological examination conducted at leastone day, week, or month after the initial exam. Improved neurologicalfunction at the subsequent exam compared to the initial exam indicatesresultant axonal regeneration. The specific detection and/or examinationmethods used will usually be based on the prevailing standard of medicalcare for the particular type of axonal lesion being evaluated (i.e.trauma, neurodegeneration, etc.).

The invention also provides activator-eluting or activator-impregnatedCNS implantable solid or semi-solid devices. Examples of CNS implantabledevices include polymeric microspheres (e.g. see Benny et al., ClinCancer Res. (2005) 11:768-76) or wafers (e.g. see Tan et al., J PharmSci. (2003) 4:773-89), biosynthetic implants used in tissue regenerationafter spinal cord injury (reviewed by Novikova et al., Curr Opin Neurol.(2003) 6:711-5), biodegradable matrices (see e.g. Dumens et al.,Neuroscience (2004) 125:591-604), biodegradable fibers (see e.g. U.S.Pat. No. 6,596,296), osmotic pumps, stents, adsorbable gelatins (seee.g. Doudet et al., Exp Neurol. (2004) 189:361-8), etc. Preferreddevices are particularly tailored, adapted, designed or designated forCNS implantation. The implantable device may contain one or moreadditional agents used to promote or facilitate neural regeneration. Forexample, in one embodiment, an implantable device used for treatment ofacute spinal cord injury contains the activator and methylprednisoloneor other anti-inflammatory agents. In another embodiment, theimplantable device contains the activator and a nerve growth factor,trophic factor, or hormone that promotes neural cell survival, growth,and/or differentiation, such as brain-derived neurotrophic factor(BDNF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF),inosine, oncomodulin, NT-3, etc.

Example 1: Promoting Axon Regeneration in the Mature CNS by PromotingProtein Translation

Germline knockout of individual cell growth control genes result ineither embryonic lethality or have compromised viability in mice, thuslimiting the utility of these animals in adult axon regenerationstudies. To circumvent this problem, we utilized a viral strategy toexpress Cre recombinase to conditionally delete specific genes in adultRGCs. To validate efficient expression of Cre in RGCs, we injectedadeno-associated viruses expressing Cre (AAV-Cre) into the vitreous bodyof the eye in reporter mice (Rosa-STOP-PLAP) in which PLAP ((humanplacenta alkaline phosphatase) activity is dependent on the expressionof Cre. We found that PLAP expression is induced in more than 90% ofRGCs, indicating that AAV-Cre is highly efficient in infecting RGCs inadult mice. The completeness of optic nerve transection was verified bythe observations that after lesion both retrograde and anterogradetracers fail to reach the retina or the superior colliculi (SC),respectively. Because lens injury and subsequent activation ofmacrophages have been reported to promote axon regeneration in vivo (9),the micropipette used for injections was angled in such a way to avoidtouching lens. We verified that this method resulted in no significantmacrophage/microglia activation after intravitreal AAV injection asindicated by anti-CD68 antibody staining.

To test the effects of conditional deletion of growth control moleculeson axon regeneration in adult RGCs, we injected AAV-Cre into thevitreous body of different adult floxed mice, including Rbf/f (12),P53f/f (12), Smad4f/f (13), Dicerf/f (14), LKB1f/f (15), and PTENf/f(16). The deletion of individual genes in RGCs was verified by in situhybridization experiments. A standard optic nerve crush assay (9, 17)was therefore performed at 2 weeks following AAV injection. Axonregeneration was assayed by examining axonal fibers labeled with theanterograde tracer, cholera toxin B (CTB), in the optic nerve sectionsacross the lesion site. Since approximately 80% of axotomized RGCs inwild type mice undergo cell death within 2 weeks post-injury (18);neuronal survival was also examined by whole-mount staining of theretina with anti-TUJ1 antibodies (a marker of RGCs).

We found that inhibiting negative regulators of growth promotingpathways promotes neuron survival and/or axon regeneration. PTENinhibition had the most dramatic effects on both neuronal survival andaxon regeneration. In all PTENf/f conditional mutants injected withAAV-Cre, but not with control AAV-GFP, RGCs displayed a significantincrease in RGC survival. In addition, striking long-distance axonregeneration was observed at 14 days post-injury. We repeated theAAV-Cre experiments in a second set of PTENf/f mice, as well as AAV-GFPinjection as controls, and observed similar results. These data indicatethat inhibition of PTEN is sufficient in enhancing regeneration of theinjured adult CNS. Quantitatively, an estimated 45% of PTEN-deleted RGCssurvived at two weeks post injury, in comparison to approximately 20% incontrol animals. Of the surviving RGCs, at 2 weeks post-injury,approximately 8-10% regenerate their lesioned axons up to 0.5 mm distalto the lesion epicenter. To our knowledge, this is the first descriptionof such long distance regeneration in the injured adult mammalian opticnerve. Importantly, these regenerating fibers continued to project alongthe optic nerve over time. At 4 weeks post-injury, some regeneratingfibers extended to the area of the optic chiasm. Among the other mouselines tested, p53-deleted RGCs showed a significant increase in neuronalviability (about 54% of injured RGCs survived) but there was no evidenceof axon regeneration in these mice. Since both P53 and PTEN deletionenhanced cell survival, while only PTEN-deletion promoted axonregeneration, these data indicate that inducing neuron survival may notbe sufficient to trigger axon regrowth (19). Our results indicate thatPTEN-inhibition acts upon intrinsic regenerative mechanisms to promotegrowth in the adult CNS following injury.

We hypothesized that PTEN-inhibited CNS axons may quickly resume axonelongation after injury. We thus performed intravitreal CTB injectionimmediately after optic nerve crush to trace axons at early time pointspost-injury. At the lesion site, an obvious glial response occurred at 1to 3 days after crushing as indicated by the up-regulation ofchondroitin sulfate proteoglycans (CSPGs) expression. Consistent withprevious studies (20), GFAP-positive astrocytes are largely excludedfrom the lesion sites and CSPG signals returned to low levels at 7 dayspost-injury. At 1 day post-lesion, injured optic nerve fibers terminateat the proximal end of the crush site both in control and in AAV-Creinjected PTENf/f mice. However, at 3 days post-injury, axonal sproutsfrom PTEN-deficient RGCs started to penetrate into the CSPG-enrichedlesion site together with macrophages and some fibers could be seenbeyond the lesion sites at 7 days after injury. In contrast, minimumaxonal sprouts were seen in control animals at these stages. Electronmicroscopic analysis confirmed that in the wild type situation,degenerating RGC axons, myelin debris and macrophages occupied injurysites, and few regenerating fibers were visible. However, when PTEN wasinhibited in RGCs, regenerative axonal sprouts, often appearing asbundles, were found both within and distal to the lesion site in earlydays post-injury. These results indicated that PTEN-inhibition indeedenabled axons to overcome inhibition in the lesion site and toregenerate soon after injury.

PTEN deletion is known to result in the activation of PI3K/mTOR pathway,which is critical in controlling cell growth and size by regulating thecap-dependent protein translation initiation (21-24). Because axonregeneration requires substantial new protein synthesis, we hypothesizedthat mTOR activation could underlie axon regrowth in PTEN-deletedneurons. PTEN and different forms of PI3 kinases are expressed in wildtype adult RGCs. Two of the well-studied targets of the mTOR kinase thatmediates its effects on protein translation are ribosomal S6 kinase 1(S6K1) and the eukaryotic initiation factor 4 E (eIF4E)-binding protein4E-BP1. Phospho-S6K1 in turn phosphorylates ribosomal protein S6.Previous studies have used the phosphorylation of S6 and 4E-BP1 asindicators of mTOR activation (25, 26). Phosphorylated-S6 (p-S6)staining revealed a dramatic development-dependent decline in thepercentage of p-S6 positive RGCs. Strong p-S6 signals can be seen inmost embryonic neurons, but remain in only a small number of adult RGCs,indicating that mTOR signaling is down-regulated in the majority ofadult RGCs. These changes correlate with the decrease in the overallaxonal growth abilities as these neurons mature.

We next sought to determine why the subset of adult RGCs with p-S6signals still cannot regenerate their injured axons. It has beenpreviously reported that upon experiencing stresses such as hypoxia,cells respond by suppressing mTOR signaling (27-30), an evolutionallyconserved stress response proposed to maintain energy homeostasis forsurvival. Thus, we postulated that stress(es) resulting from axotomy mayreduce global translation in injured neurons. To test this, we estimatedthe rate of new protein synthesis in purified RGCs from control andinjured rats. Control and injured RGCs were incubated with35S-methanine/cysteine and the extracts from these cells were resolvedusing SDS-PAGE. 35S-methanine/cysteine incorporated proteins normalizedagainst total protein contents were quantified, and the results showed asignificant decrease in new protein synthesis in axotomized adult RGCs.

We next studied whether nerve lesion down-regulates the activity of themTOR pathway by immunohistochemical analysis of retinal sections withanti-p-S6 antibodies. Our results indicate that axotomy almostcompletely abolished the remaining p-S6 signals in adult RGCs at allpost-injury time points examined (1, 3, 7 days) in control mice,indicating that axotomy triggers a rapid and sustained down-regulationof mTOR mediated signals, and that antagonizing this down-regulationpromotes regeneration. in wild type adult CNS neurons.

Previous work suggested that the up-regulation of stressed inducedmolecules Redd1/2 (regulated in development and DNA damage responses1/2) may mediate the inhibition of mTOR in cells under stress (27-30).We thus analyzed Redd1/2 expression in injured neurons by quantitativereal time PCR (q-PCR) using mRNAs derived from FACS-purified rat RGCs.No significant changes in Redd1/2 mRNA were found in axotomized RGCs. Asa positive control, we found that the expression of Gadd45a, a geneknown to be up-regulated after axotomy (31), is significantly increasedin this same set of injury samples.

We next tested whether PTEN inhibition affects the p-S6 level in theadult RGCs both before and after injury. In the uninjured AAV-Creinjected PTENf/f mice, the percentage of anti-phospho-S6 stained RGCswas not significantly different from that in wild type or in AAV-GFPinjected PTENf/f mice. However, the intensity of p-S6 signal appearedenhanced and these p-S6 positive RGCs showed increased cell size,consistent with previous studies of PTEN-deletion in other types ofneurons (32, 33). Importantly, after optic nerve crush, similarpercentages of p-S6 positive RGCs remained at 1, 3, or 7 dayspost-injury in the AAV-Cre injected PTENf/f mice. Thus, despite a stressresponse to axotomy, these axotomized neurons with PTEN deletion stillpossess the mTOR activity at the levels similar to uninjured wild typeneurons. The percentage of regenerating fibers (8-10%) was similar tothat of p-S6 positive axotomized RGCs (8-10%).

To further examine whether activation of the mTOR pathway is sufficientto promote axon regeneration, we performed similar optic nerveinjury/regeneration assays using TSC1f/f conditional knockout mice (34).TSC1 and 2 form a protein complex which negatively regulates mTORsignaling (35-39). Previous reports indicated that loss of either TSC1or TSC2 leads to constitutive activation of mTOR pathway (38). Asexpected, in AAV-Cre injected TSC1f/f mice, strong p-S6 signals wereobserved in axotomized RGCs, and there was a significant enhancement ofRGC survival after injury. More importantly, considerable axonregeneration was observed in TSC1-deleted but not in wild type miceinjected with AAV-Cre. The extent of axon regeneration in TSC1 deletedmice was slightly weaker than that induced by PTEN deletion, indicatingthat changes in other downstream targets of PTEN, such as Akt and GSK-3activity (40, 41), may also be involved in regenerative growth.Nonetheless, these results indicate that activating the mTOR pathway issufficient to promote both CNS neuron survival and axon regeneration.

Our results reveal that mTOR activity is suppressed in axotomized CNSneurons by a two-step down-regulation of the mTOR signaling; first bydevelopmental maturation and second by axotomy-triggered stressresponse. Our results provide a new avenue to promote long distance CNSaxon regeneration after injury, wherein increasing positive regulatorsof mTOR signaling, or inhibiting negative regulators of mTOR signaling(such as with chemical inhibitors of TSC1/2 or PTEN (42)), may be usedtransiently after CNS injury to prevent the down-regulation of proteinsynthesis and to promote axon regeneration and functional recovery.

Example 2a: Pharmacological PTEN Inhibition Promotes Regeneration ofLesioned Optic Nerve Fibers in Adult Mice

We designed a similar optic nerve study to demonstrate thatpharmacological inhibitors of neuronal PTEN activity similarly promoteaxon regeneration, adapting a previously described model of optic nervecrushing [Fischer et al, J. Neurosci. 18, 1646 (2004)]. Adult mouseoptic nerves are exposed behind the eyeball and crushed. Immediatelyafter injury in adult mice, GELFOAM™ soaked in solutions of alternativePTEN inhibitors at serial concentrations (Table 1) or 0.1% DMSO(control) is placed against the crush site of the nerve and replacedevery three days for the first six days of the study.

TABLE 1 PTEN inhibitor solutions PTEN Inhibitor Concentrations potassiumbisperoxo(bipyridine)oxovanadate 10, 100, 1,000 ng/ml (V) (bpV(bipy))dipotassium bisperoxo(5-hydroxypyridine- 10, 100, 1,000 ng/ml2-carboxyl)oxovanadate (V) (bpV(HOpic)) potassiumbisperoxo(1,10-phenanthroline) 10, 100, 1,000 ng/ml oxovanadate(V),(bpV(phen)) dipotassium bisperoxo(picolinato)oxovanadate 10, 100,1,000 ng/ml (V), (bpV(pic))

Animals are sacrificed two weeks post injury followed by transcardialperfusion with 4% paraformaldehyde. Optic nerves are cryosectioned at 10μm and stained with an anti-GAP43 antibody (Chemicon) to detectregenerating axons [Fischer et al, supra]. Little regeneration isdetected in DMSO-treated control mice. However, injury site applicationof PTEN inhibitors results in significant increases in axonal regrowthand the number of regenerating axons, measured 0.25 mm beyond the injurysite, compared to control mice.

Example 2b: Pharmacological PTEN Inhibition Promotes Regeneration ofLesioned Optic Nerve Fibers in Adult Mice

In subsequent similar experiments, we injected PTEN inhibitors of Table2 into the retina, performed optic nerve injury, and found increasedneuronal survival and axon regeneration.

TABLE 2 PTEN inhibitors

  pTEN IC50 = 5 uM

  pTEN IC50 = 2 uM

  pTEN IC50 = 0.3 uM

Example 3: Exemplary PTEN Inhibition Assays for General Screening andIC50 Determinations

PTEN inhibitors are evaluated in an inhibition assay conducted inhalf-volume 96 well plates in 25 ul total volume per well containing 2mM dithiothreitol (DTT) and 0.1 mM Tris buffer, pH 8.0 and up to 3 ugtotal protein of PTEN. Small volumes of the test inhibitor candidates(stock concentrated solutions of 25 mM in DMSO) are mixed with the PTENsolution at room temperature for about 10 minutes and then substrate isadded. The reaction mix is then incubated in 37° C. for 20 minutes.Subsequent to this a 100 ul aliquot of malachite green buffer (Upstate,Charlottesville, Va.) is added to develop the color in the dark at roomtemperature (this solution also stops the dephosphorylation reaction). ASpectraMax Plus spectrophotometric plate reader (Molecular Devices,Sunnyvale, Calif.) is used to measure the optical density at 650nanometers.

The initial screening concentration of inhibitor candidates is 250 uMand candidates with inhibition greater than 50% compared with ano-inhibitor control group are then evaluated further to determine IC50values. PTEN can be purchased commercially or prepared by literaturemethods [i.e. from cell extracts of bacteria expressing geneticreconstituted Glutathione-5-transferase (GST)-PTEN fusion proteinwhereupon the GST-PTEN in the cell extract is bound onto and purifiedfrom Glutathione Sepharose 4B gel (Amersham, Piscataway, N.J.)].Suitable PTEN reaction substrates include (a) PIP3 Phospholipid vesicle(PLV), which may be made using published methods (Maehama et al. 2000,Analytical Biochemistry 279, 248-250) and is typically utilized at about50 uM in the final reaction mixture (based on component concentration),(b) water soluble PIP3 Echelon Biosciences, Salt Lake City, Utah,utilized at a working concentration of 100 uM, and (c) phosphorylatedpoly glutamic-tyrosine peptide designated (EEEEYp)_(n), where n=2 or 3(Biofacilities of Indiana University, Indianapolis, Ind.), wherein aworking concentration of the phosphorylated tyrosine substrate is 200uM.

To determine the dose response of potential PTEN inhibitors, doses oftest compounds ranging from 1 nM to 250 uM (final reaction mixconcentrations) are evaluated in the general PTEN inhibition assay(supra). To obtain performed IC50 data, two separate rounds of the doseresponse assay are performed. In the first round, PTEN activity istested in the presence of inhibitor at 10 fold serial dilutions rangingfrom 1 nM to 250 uM. Once the concentration range is determined, atwhich PTEN activity changes dramatically, two additional concentrationdata points within this range are added and the PTEN inhibition assay isthen rerun for the second round. The PTEN inhibition IC50 is presentedas the inhibitor concentration at which 50% of the PTEN activity(measured by phosphate production and compared to un-inhibited controlsamples) is found. The IC50 determination from the data is made usingPrism software (GraphPad Software, San Diego, Calif.).

Example 4: Determination of PTEN Inhibition on Axonal Regeneration afterSpinal Injury in Rats

This animal study demonstrates that in an animal model for spinalinjury, axonal regeneration can be promoted by intrathecal orintravenous administration of PTEN inhibitors bpV(bipy), bpV(HOpic),bpV(phen) and bpV(pic). Methodology for this animal study was adaptedfrom Nash et al (J. Neurosci (2002) 22:7111-7120), Luo et al (MolecularPain (2005) 1:29), and Obata et al (J. Neurosci. (2004) 24:10211-22).

Adult Sprague Dawley rats (300-400 μm) are trained and tested in adirected forepaw reaching (DFR) apparatus which measures graspingability. The apparatus, which is described in detail by Nash et al.,supra, is a box that consists of two compartments: a main compartmentfor housing the rats and a minor compartment for the food, separated bya PLEXIGLAS™ divider. The minor compartment is subdivided into slots ofequal size, each holding a pellet of food. Between the slots and thePLEXIGLAS™ there is a gap. The apparatus is configured such that inorder to retrieve a food pellet from a slot, a rat must extend aforelimb through a hole in the PLEXIGLAS™ divider, and grasp the pelletand lift it over the gap and out of the slot. If the rat merely rakesthe food in the slot towards the hole in the PLEXIGLAS™, the food willdrop from the slot into the gap and fall to the floor of the minorcompartment. The floor of the minor compartment can be configured toallow a rat to retrieve food that drops or it can be lowered to preventthe rat from reaching dropped food. Prior to inducing spinal injury, therats are food restricted, receiving ˜3 μm food/100 μm body weight perday, before and throughout training and testing. Weight is monitored toensure that rats are reduced to no less than 80% of their original bodyweight at any time. All rats are given shaping periods for 2-3 d in thebox to allow them to learn the task while they become familiar with thetesting situation. Animals are trained twice per day for 5 d and thentested twice per day for 5 d, and presurgical DFR data is collected.During the testing period, rats are given 5 min to complete the task andare allowed to make as many attempts as they want during this timeperiod. Rats are required to return to at least 95% of their originalweight to ensure that they are healthy before undergoing surgery.

Rats are randomly assigned to control or experimental groups. Shamcontrol rats undergo surgical procedure without lesioning, and with orwithout placement of a mini osmotic pump (Alzet type 2001; Durect,Cupertino, Calif.). Lesioned control rats receive no treatment, tailvein injection with vehicle only treatment, insertion of a mini osmoticpump only treatment, or insertion of a mini osmotic pump with vehicleonly treatment. After anesthesia with isoflurane, the rats are placed onan operating board in such a way as to bend the cervical spinal cord formaximum exposure. A laminectomy is performed exposing the dorsum of thespinal cord between C2 and C4. The dorsal columns are identifiedbilaterally, and, in all rats except for those in the sham group, asuture needle is passed through the spinal cord, isolating the dorsalfuniculus. The suture thread is gently lifted, and a pair of iridectomyscissors is used to bilaterally transect the dorsal funiculus, therebytransecting the dorsal corticospinal tract (CST). Visualization of thedorsal horns and the central gray commissure confirms accuracy of thelesion borders. A pledget of biodegradable GELFOAM™ soaked in afluorescent retrograde tracer, FLUOROGOLD™ (3% in 0.9% saline; MolecularProbes), is placed in the lesion site to identify the neurons whoseaxons are transected, confirming the lesion. Rats designated for PTENinhibitor treatment or corresponding control treatment are implantedwith mini osmotic pumps adjacent to the lesion site. The pumps in thetreatment group operate at a rate of 1 μl/hr for a period of 7 days andare filled with PTEN inhibitor (bpV(bipy), bpV(HOpic), bpV(phen) orbpV(pic)) at a concentration of 500 ng/μ1. The overlying muscles andskin are sutured, and the rats are placed on a heating pad to maintainbody temperature. Each rat receives a single dose of buprenorphine (0.1mg/kg) immediately after surgery to alleviate pain.

One hour after the spinal cord is lesioned, the rats in the tail veininjection treatment group receive a bolus injection of 100 μg/kg PTENinhibitor (bpV(bipy), bpV(HOpic), bpV(phen) or bpV(pic)) in asaline/DMSO vehicle. The treatment is repeated every 24 hours on days 1through 7 post-lesion. Vehicle only control rats undergo the sametreatment but are injected with an equal volume of saline/DMSO in a tailvein.

Rats are trained twice per week during weeks 2-5 after surgery. Somerats may be profoundly impaired such that they may not be able to graspfood in the DFR task in the early postsurgical period. In this case, theapparatus can be configured to allow the rats to rake food into the maincompartment that drops from the slot onto the floor of the minorcompartment (see Nash et al., supra). This ensures that the reachingportion of the DFR task does not extinguish. The severity of thegrasping impairment decreases as the postsurgical period increases, andthe configuration of the apparatus that does not permit food raking canbe gradually reestablished. By the end of the postsurgical recoveryperiod, all rats are able to successfully perform the DFR task, to somedegree. During the sixth week after surgery, rats are tested twice perday for 5 d, and postsurgical DFR data is collected by a blindedinvestigator. Just as during the presurgery testing period, the rats areallowed 5 min to complete the task during the postsurgery testing periodand are allowed to make as many attempts as they want during this timeperiod. The data is collected in terms of total number of attempts andpercentage of successful attempts. An attempt is scored only when a ratreaches into a slot and displaces the pellet or drops it to the floor ofthe minor compartment. A successful attempt is scored when a rat graspsa pellet, lifts it over the gap and pulls it through the PLEXIGLAS™divider into the main portion of the testing apparatus.

Sham animals perform the DFR task as well postsurgically as they dopresurgically, demonstrating that only the lesion, and no other portionof the surgical procedure, inhibits the rats' abilities to perform theDFR task. The lesion and vehicle groups are the most impaired of all ofthe groups after surgery. The lesion and vehicle groups are able toperform the DFR task with a success rate of only about 40%.Significantly better performance by the SP600125-treated groupdemonstrates the effect of the treatment on functional recovery afterspinal injury.

Seven weeks after injury, rats are prepared for injection of biotindextran tetramethylrhodamine (BDT; Molecular Probes). This fluorescentanterograde tracer, injected into the primary motor cortex, is used tolabel CST axons caudal to the lesion site in the spinal cord. Afteranesthesia with isoflurane (5%), rats are placed in a stereotaxicinstrument, and a total of six stereotaxically determined holes (0.9 mmdiameter) are drilled in the skull over the primary motor corticesassociated with the forelimbs. The anteroposterior (AP) and mediolateral(ML) coordinates for these injections, from bregma, are as follows: ±0.5AP and ±3.5 mL; ±1.5 A/P and ±2.5 mL; and ±2.5 AP and ±1.5 mL. Allinjections are delivered at a depth of 2.5 mm from the surface of theskull. A 10 μl Hamilton syringe is used to inject BDT bilaterally intolayer V of the cortex. Three injections into each cortical hemisphereare used to administer a total of 1.2 μl of the anterograde tracer. Bonewax (Ethicon, Somerville, N.J.) is used to seal the holes in the skull,the scalp is sutured, and a single dose of buprenorphine (0.1 mg/kg) isadministered immediately after surgery to alleviate pain. Rats arekilled 3 d after tracer injections.

Seven weeks and 3 d after lesioning, rats are anesthetized with chloralhydrate (10 ml/kg) and perfused transcardially with 300 ml of PBS, pH7.4, followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphatebuffer. After the animals are killed, all brains and spinal cords areremoved and soaked overnight in 30% sucrose in a 0.1M phosphate buffersolution. The brains are cut coronally and the spinal cords are cuthorizontally at a thickness of 20 μm with a freezing microtome andmounted on PROBEON™ (Fisher Scientific, Pittsburgh, Pa.) coated slides.Brain and spinal cord sections are examined using a Nikon (Tokyo, Japan)Labophot fluorescent microscope, and images are captured using a digitalstill camera. The forelimb representation of the primary motor cortex isidentified based on the stereotaxic BDT injection sites. The primarymotor cortex is examined in all rats. Presence of FLUOROGOLD™-labeledneurons in layer V of the primary motor cortex, confirms that the dorsalCST axons were transected during the lesioning procedure. Because allCST axons located in the dorsal funiculus are transected during surgeryand not just those in the forelimb representation, FLUOROGOLD™-labeledneurons are found throughout the primary motor cortex in layer V. Theonly exception to this labeling pattern is in the brains of the rats inthe sham group whose brains have no FLUOROGOLD™ label.

The spinal cord caudal to the lesion is examined, and the BDT-labeledaxons occupying the region of the spinal cord normally occupied by thedorsal CST are counted. For each section, the number of BDT-labeledaxons is counted at 3 mm intervals caudal to the lesion, beginning 1 mmdistal to the injury (i.e., 1 mm, 4 mm, 7 mm, etc.) and ending 19 mmcaudal to the lesion site. Innervation of the rat forepaw extends to T1,a distance of 15.1 mm from the lesion at C3. Therefore, analysis of theaxons out to 19 mm caudal to the lesion ensures that the entire distancerepresenting the forepaw is examined. At each interval, the total numberof BDT-labeled axons (left and right CST combined) along a 500 μm length(length of microscope field) is counted. In each field counted, thefocal plane is adjusted up and down to ensure that a single continuousaxon is not double counted if it traverses out of the focal plane andreemerges farther down in the same field. The number of BDT-labeledaxons present is examined for control and experimental groups at each ofthe distances (i.e. 1, 4, 7 . . . and 19 mm caudal to the lesion site).Throughout all of the examined intervals, the mean number of axons ishighest in the sham group, and, at each distance examined, the meannumber of labeled axons in the sham group is significantly higher thanin the other groups. No significant difference is observed between themeans of the lesion and vehicle groups at any distance examined. Inthese groups, axons are found only a short distance caudal to theinjury, and, by 10 mm distal to the lesion to the farthest distanceexamined, all of the tissue is virtually devoid of axons. Significantlymore labeled axons at each distance in the PTEN inhibitor-treated groupcompared to lesioned control rats demonstrates that this treatmentpromotes axonal regeneration after spinal injury.

Example 5: Determination of Neurological Outcome Following PTENInhibitor Treatment for Acute Spinal Cord Injury

We adapted our protocol for this study from the Sygen® Multicenter AcuteSpinal Cord Injury Study described by Geisler et al (Spine (2001)26:587-598). It is a prospective, double-blind, randomized, andstratified multicenter trial, randomizing approximately 800 patients soas to have at least 720 completed and evaluable in each treatment group:placebo, low-dose PTEN inhibitor (bpV(bipy), bpV(HOpic), bpV(phen) orbpV(pic)), and high-dose PTEN inhibitor. The patients are stratifiedinto six groups, according to three degrees of injury severity (AmericanSpinal Injury Association grades A, B, and C+D) and two levels ofanatomic injury (cervical and thoracic). The trial is sequential withpreplanned interim analyses as each group of 720/4=180 patients reachtheir 26-week examination and become evaluable. Patients are required tohave at least one lower extremity with a substantial motor deficit.Patients with spinal cord transection or penetration are excluded, asare patients with a significant cauda equina, brachial or lumbosacralplexus, or peripheral nerve injury. Gunshot injuries that do notpenetrate the cord are allowed. Multiple trauma is allowed as long as itis not so severe as to prevent neurologic measurement evaluation orinterpretation.

All patients are to receive the second National Acute Spinal Cord InjuryStudies (NASCIS II) dose regimen of methylprednisolone (MPSS) startingwithin 8 hours after the spinal cord injury (SCI). To avoid any possibleuntoward interaction between MPSS and PTEN inhibitors the studymedication is not started until after completion of MPSS administration.

The placebo group has a loading dose of placebo and then 56 days ofplacebo. The low dose PTEN inhibitor group has a 50-mg loading doseadministered intravenously (i.v.) followed by 10 mg/day i.v. for 56days. The high dose PTEN inhibitor group has a 250-mg loading dosefollowed by 50 mg/day for 56 days.

The baseline neurologic assessment includes both the AIS and detailedAmerican Spinal Injury Association (ASIA) motor and sensoryexaminations. Modified Benzel Classification and the ASIA motor andsensory examinations are performed at 4, 8, 16, 26, and 52 weeks afterinjury. The Modified Benzel Classification is used for post-baselinemeasurement because it rates walking ability and, in effect, subdividesthe broad D category of the AIS. Because most patients have an unstablespinal fracture at baseline, it is not possible to assess walkingability at that time; hence the use of different baseline and follow-upscales. Marked recovery is defined as at least a two-grade equivalentimprovement in the Modified Benzel Classification from the baseline AIS.The primary efficacy assessment is the proportion of patients withmarked recovery at week 26. The secondary efficacy assessments includethe time course of marked recovery and other established measures ofspinal cord function (the ASIA motor and sensory scores, relative andabsolute sensory levels of impairment, and assessments of bladder andbowel function).

Example 6: Determination of Effect of PTEN Inhibition after CorticalImpact Injury in Rats

We adapted methodology from Cherian et al. (J Pharmacol Exp Ther. (2003)304:617-23), to test the effects of different doses and treatmentschedules of PTEN inhibitors on a rat model of brain impact injury. Atotal of 60 male Evans rats weighing 300 to 400 g are assigned to one ofthe following doses injected intraperitoneally (i.p.) or intracerebralventricularly (i.c.v.): none (saline control group), 0.01, 0.1, 1.0, and10.0 mg/kg/day PTEN inhibitors (bpV(bipy), bpV(HOpic), bpV(phen) orbpV(pic)). The rats are further assigned to a treatment duration of 1,3, 7, or 14 days, with 4 rats in each treatment group, and 3 rats ineach control group (i.e. saline administered for 1, 3, 7, or 14 days).

The details of the methods to produce the impact injury have beenpreviously described (Cherian et al., J. Neurotrauma (1996) 13:371-383).Briefly, the head of the rat is fixed in a stereotaxic frame by ear barsand incisor bar. A 10-mm diameter craniotomy is performed on the rightside of the skull over the parietal cortex. An impactor tip having adiameter of 8 mm is centered in the craniotomy site perpendicular to theexposed surface of the brain at an angle of approximately 45 degrees tothe vertical. The tip is lowered until it just touches the duralsurface. The impactor rod is then retracted, and the tip advanced anadditional 3 mm to produce a brain deformation of 3 mm during theimpact. Gas pressure applied to the impactor is adjusted to 150 psi,giving an impact velocity of approximately 5 m/s and duration ofapproximately 150 to 160 ms.

Rats are fasted overnight and anesthetized with 3.5% isoflurane in 100%oxygen in a vented anesthesia chamber. Following endotracheal intubationwith a 16-gauge Teflon catheter, the rats are mechanically ventilatedwith 2% isoflurane in 100% oxygen for the surgical preparation and forthe impact injury. Intracranial pressure (ICP) is monitored by a 3Fmicrosensor transducer (Codman & Schurtleff, Randolph, Mass.) insertedin the left frontal lobe, well away from the impact site. ICP ismonitored during the impact injury as a measure of the severity of theinjury. Rectal temperature is maintained at 36.5-37.5° C. by a heatingpad, which is controlled by rectal thermistor. Brain temperature is keptconstant at 37° C. with the help of a heating lamp directed at the head.

The rats that are to receive i.c.v. administration of PTEN inhibitorsreceive mini-pump implants using procedures described by Kitamura et al(J Pharmacol Sci (2006) 100:142-148). Briefly, the rats are fixed in astereotaxic frame (David Kopf Instruments, Tujunja, Calif.). Guidecannulae are implanted into the left lateral ventricle (Bregma −0.8 mm,lateral 1.5 with a depth of 3.7 mm below the dura). Each cannula is thenconnected by a catheter to an ALZET® mini-osmotic pump implantedsubcutaneously in the scapular region and configured to continuouslyinfuse the drug to achieve the specified daily dose of AS601245 (orvehicle only for control groups).

For rats in the i.p. treatment group, each dose of PTEN inhibitor isdissolved in 1 ml of sterile 0.9% saline so that the volume delivered isthe same for each group and only the dosage of PTEN inhibitor varies.The first dose is administered within 1 hour following impact injury.And once daily thereafter for the assigned treatment duration.

After removing all catheters and suturing the surgical wounds, the ratsare allowed to awaken from anesthesia. For the first 3 days post injury,the rats are treated with butorphanol tartrate, 0.05 mg of i.m. every 12h (twice a day), for analgesia and enrofloxacin 2.27%, 0.1 ml of IM qd,to reduce the risk of postoperative infections.

The outcome measures are performed by investigators who are blinded tothe treatment group. At 2 weeks after the impact, the animals are deeplyanesthetized with a combination of ketamine/xylazine/acepromazine andperfused transcardially with 0.9% saline, followed by 10% phosphatebuffered formaldehyde. The entire brain is removed and fixed in 4%formalin. The fixed brains are examined grossly for the presence ofcontusion, hematoma, and herniation. The brains are photographed,sectioned at 2-mm intervals, and then embedded in paraffin. Hematoxylinand eosin (H&E) stained 9-μm thick sections are prepared for histologicexamination. Particular care is made to include the largestcross-sectional area of cortical injury on the cut surface of theembedded sections. The H&E-stained coronal sections are digitized usinga POLAROID™ Sprint Scanner (POLAROID™ Corporation, Waltham, Mass.)equipped with a PATHSCAN™ Enabler (Meyer Instruments, Houston, Tex.).The injury volume is measured by determining the cross-sectional area ofinjury in each H&E-stained coronal image and multiplying by thethickness of the tissue between the slices. This slab volume techniqueis implemented on the image processing program Optimas 5.2 (OptimasCorporation, Seattle, Wash.). Neurons in the middle 1-mm segments of theCA1 and CA3 regions of the hippocampus are counted at a magnification of200×. Neurons are identified by nuclear and cytoplasmic morphology, andindividual cells are counted whether normal or damaged. Neurons withcytoplasmic shrinkage, basophilia, or eosinophilia or with loss ofnuclear detail are regarded as damaged. The regions measured are 1 mmlong and 1 mm wide (0.5 mm on either side of the long axis of thesegment). The total number of neurons and the number of neurons thatappear normal are expressed as neurons per squared millimeter.

Example 7: Determination of Whether PTEN Inhibition Promotes NeuralRegeneration in Animal Models of Focal Brain Ischemia

This study uses previously described methods (Brines et al, Proc NatlAcad Sci USA. (2000) 97:10526-31) to demonstrate the effect ofsystemically administered PTEN inhibitors in an animal model of focalbrain ischemia. Sprague-Dawley male rats weighing ˜250 g areanesthetized with pentobarbital [60 mg/kg body weight (BW)]. Body coretemperature is thermostatically maintained at 37° C. by using a waterblanket and a rectal thermistor (Harvard Apparatus) for the duration ofthe anesthesia. The carotid arteries are visualized, and the rightcarotid is occluded by two sutures and cut. A burr hole adjacent androstral to the right orbit allows visualization of the MCA, which iscauterized distal to the rhinal artery. Animals are then positioned on astereotaxic frame. To produce a penumbra surrounding this fixed MCAlesion, the contralateral carotid artery is occluded for 1 h by usingtraction provided by a fine forceps. 0.5 ml of a 1 μg/ml solution ofPTEN inhibitor (bpV(bipy), bpV(HOpic), bpV(phen) or bpV(pic)) or vehiclecontrol is administered at 1 hr, 1 day, 5 days, or 10 days from theonset of the reversible carotid occlusion. To evaluate the extent ofinjury, the animals are killed after 15 days, the brains are removed,and serial 1-mm thick sections through the entire brain are cut by usinga brain matrix device (Harvard Apparatus). Each section is thenincubated in a solution of 2% triphenyltetrazolium chloride (wt/vol) in154 mM NaCl for 30 min at 37° C. and stored in 4% paraformaldehyde untilanalysis. Quantification of the extent of injury is determined by usinga computerized image analysis system (MCID, Imaging Research, St.Catharine's, ON, Canada). To accomplish this, a digital image of eachsection is obtained and the area of injury delineated by outlining theregion in which the tetrazolium salt is not reduced, i.e., nonviabletissue. For cases in which the necrosis is so severe that tissue isactually lost and therefore the borders can not be directly assessed, anoutline of the contralateral side is used to estimate the volume ofinjured brain. Total volume of infarct is calculated by reconstructionof the serial 1-mm thick sections.

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The foregoing examples and detailed description are offered by way ofillustration and not by way of limitation. All publications and patentapplications cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims

What is claimed is:
 1. A method for promoting survival of, and axonregeneration in a lesioned mature human neuron in vivo, wherein thelesion results from a traumatic spinal cord injury arising from physicalforce, comprising contacting the lesioned neuron with a therapeuticallyeffective amount of a PTEN specific inhibitor to thereby promotesurvival of, and axon regeneration in the neuron.
 2. The method of claim1, further comprising detecting the resultant promotion of the survivalof, and axon regeneration in the neuron.
 3. The method of claim 2,wherein the detecting step is by an indirect assay of axon regeneration.4. The method of claim 2, wherein the detecting step is by a directassay of axon regeneration.
 5. The method of claim 1, wherein theinhibitor is administered locally at the neuron.
 6. A method forpromoting axon regeneration in a lesioned mature human central nervoussystem (CNS) neuron in vivo, wherein the lesion results from a traumaticspinal cord injury arising from physical force, comprising contactingthe neuron with a therapeutically effective amount of a PTEN specificinhibitor to thereby promote axon regeneration in the neuron.
 7. Themethod of claim 6, wherein the neuron is in the spinal cord.
 8. Themethod of claim 6, wherein the inhibitor is administered locally at theneuron.
 9. A method for promoting axon regeneration in a lesioned maturehuman neuron in vivo, wherein the lesion results from acute spinal cordinjury arising from physical force, comprising contacting the lesionedneuron with a therapeutically effective amount of a PTEN specificinhibitor to thereby promote axon regeneration in the neuron.
 10. Themethod of claim 9, wherein the inhibitor is administered locally at theneuron.